Engineered enzyme

ABSTRACT

The present disclosure provides a novel catalytically inactive MAD7 nuclease (dMAD7) that retains the ability to bind DNA in a sequence-specific manner. The MAD7 nuclease from which the dMAD7 has been derived was isolated from Eubacterium rectale.

RELATED CASES

The present application is a continuation-in-part of U.S. Ser. No.16/953,253, entitled “Engineered Enzymes,” filed 19 Nov. 2020; which isa continuation of U.S. Ser. No. 16/844,079, filed 09 Apr. 2020, now U.S.Pat. No. 10,876,102; which is a continuation of U.S. Ser. No.16/798,315, filed 22 Feb. 2020, now U.S. Pat. No. 10,640,754; which is acontinuation of U.S. Ser. No. 16/658,948, filed 21 Oct. 2019, now U.S.Pat. No. 10,604,746; which claims priority to U.S. ProvisionalApplication No. 62/748,668, filed 22 Oct. 2018.

FIELD OF THE INVENTION

This invention relates to a catalytically in active MAD7 variant derivedfrom the bacterium Eubacterium rectale.

INCORPORATION BY REFERENCE

Submitted with the present application is an electronically filedsequence listing via EFS-Web as an ASCII formatted sequence listing,entitled “INSCO29CIP_seqlist_20210713”, created Jul. 13, 2021, and22,000 bytes in size. The sequence listing is part of the presentspecification and is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that themethods referenced herein do not constitute prior art under theapplicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowmanipulation of gene sequence, hence gene function. These nucleasesinclude nucleic acid-guided nucleases. The range of target sequencesthat nucleic acid-guided nucleases can recognize, however, isconstrained by the need for a specific PAM to be located near thedesired target sequence. PAMs are short nucleotide sequences recognizedby a gRNA/nuclease complex where this complex directs editing of thetarget sequence. The precise PAM sequence and pam length requirementsfor different nucleic acid-guided nucleases vary; however, PAMstypically are 2-7 base-pair sequences adjacent or in proximity to thetarget sequence and, depending on the nuclease, can be 5′ or 3′ to thetarget sequence. Screening the natural diversity of nucleic acid-guidednucleases that exist across species may allow for the discovery ofenzymes with enhanced nuclease activity or increased cleavage fidelitywhen used in a given organism; both changes that may increase theversatility of a nucleic acid-guided nuclease for certain editing tasks.

There is a need in the art of nucleic acid-guided nuclease gene editingfor catalytically inactive nucleases with which retain the ability tobind DNA in a sequence-specific manner. The novel dMAD7 enzyme derivedfrom the bacterium Eubacterium rectale described herein satisfies thisneed.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides a novel catalytically inactive MAD7nuclease (dMAD7) that retains the ability to bind DNA in asequence-specific manner. The MAD7 nuclease from which the dMAD7 hasbeen derived was isolated from Eubacterium rectale (SEQ ID NO: 1) (see,e.g., U.S. Pat. Nos. 9,982,279 and 10,337,028). dMAD7 (SEQ ID NO: 1)differs from MAD7 (SEQ ID NO: 1) by a single amino acid D877A.

Thus, in one embodiment there is provided a catalytically inactive MAD7enzyme derived from the bacterium Eubacterium rectale (dMAD7) having theamino acid sequence of SEQ ID NO: 2. In some aspects, the dMAD7 binds toa bacterial genome. In other aspects, the dMAD7 binds to a yeast genome.In yet other aspects, the dMAD7 binds to the genome of mammalian cells.

These aspects and other features and advantages of the invention aredescribed below in more detail.

DETAILED DESCRIPTION

The description set forth below in connection with the appended drawingsis intended to be a description of various, illustrative embodiments ofthe disclosed subject matter. Specific features and functionalities aredescribed in connection with each illustrative embodiment; however, itwill be apparent to those skilled in the art that the disclosedembodiments may be practiced without each of those specific features andfunctionalities. Moreover, all of the functionalities described inconnection with one embodiment are intended to be applicable to theadditional embodiments described herein except where expressly stated orwhere the feature or function is incompatible with the additionalembodiments. For example, where a given feature or function is expresslydescribed in connection with one embodiment but not expressly mentionedin connection with an alternative embodiment, it should be understoodthat the feature or function may be deployed, utilized, or implementedin connection with the alternative embodiment unless the feature orfunction is incompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, biological emulsion generation,and sequencing technology, which are within the skill of those whopractice in the art. Such conventional techniques include polymer arraysynthesis, hybridization and ligation of polynucleotides, and detectionof hybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds. (1999), Genome Analysis:A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds.(2007), Genetic Variation: A Laboratory Manual; Dieffenbach, Dveksler,Eds. (2003), PCR Primer: A Laboratory Manual; Bowtell and Sambrook(2003), DNA Microarrays: A Molecular Cloning Manual; Mount (2004),Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell(2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual;and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual(all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995)Biochemistry (4th Ed.) W. H. Freeman, New York N.Y.; Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y.; Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y.; Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, eds., John Wiley & Sons 1998); Mammalian ChromosomeEngineering—Methods and Protocols (G. Hadlaczky, ed., Humana Press2011); Essential Stem Cell Methods, (Lanza and Klimanskaya, eds.,Academic Press 2011), all of which are herein incorporated in theirentirety by reference for all purposes. Nuclease-specific techniques canbe found in, e.g., Genome Editing and Engineering From TALENs andCRISPRs to Molecular Surgery, Appasani and Church, 2018; and CRISPR:Methods and Protocols, Lindgren and Charpentier, 2015; both of which areherein incorporated in their entirety by reference for all purposes.Basic methods for enzyme engineering may be found in, Enzyme EngineeringMethods and Protocols, Samuelson, ed., 2013; Protein Engineering,Kaumaya, ed., (2012); and Kaur and Sharma, “Directed Evolution: AnApproach to Engineer Enzymes”, Crit. Rev. Biotechnology, 26: 165-69(2006).

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an oligonucleotide”refers to one or more oligonucleotides, and reference to “an automatedsystem” includes reference to equivalent steps and methods for use withthe system known to those skilled in the art, and so forth.Additionally, it is to be understood that terms such as “left,” “right,”“top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,”“upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may beused herein merely describe points of reference and do not necessarilylimit embodiments of the present disclosure to any particularorientation or configuration. Furthermore, terms such as “first,”“second,” “third,” etc., merely identify one of a number of portions,components, steps, operations, functions, and/or points of reference asdisclosed herein, and likewise do not necessarily limit embodiments ofthe present disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, methods and cell populations that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of ordinary skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TAGCTG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus by homologousrecombination using nucleic acid-guided nucleases. For homology-directedrepair, the donor DNA must have sufficient homology to the regionsflanking the “cut site” or site to be edited in the genomic targetsequence. The length of the homology arm(s) will depend on, e.g., thetype and size of the modification being made. In many instances andpreferably, the donor DNA will have two regions of sequence homology(e.g., two homology arms) to the genomic target locus. Preferably, an“insert” region or “DNA sequence modification” region-the nucleic acidmodification that one desires to be introduced into a genome targetlocus in a cell-will be located between two regions of homology. The DNAsequence modification may change one or more bases of the target genomicDNA sequence at one specific site or multiple specific sites. A changemay include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 300, 400, or 500 or more base pairs of the targetsequence. A deletion or insertion may be a deletion or insertion of 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or500 or more base pairs of the target sequence.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” or “crRNA” referto a polynucleotide comprising 1) a guide sequence capable ofhybridizing to a genomic target locus, and 2) a scaffold sequencecapable of interacting or complexing with a nucleic acid-guidednuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible and, in someembodiments—particularly many embodiments in which selection isemployed—the transcription of at least one component of the nucleicacid-guided nuclease editing system is under the control of an induciblepromoter.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin,bleomycin, streptomycin, rhamnose, puromycin, hygromycin, blasticidin,and G418 may be employed. In other embodiments, selectable markersinclude, but are not limited to human nerve growth factor receptor(detected with a MAb, such as described in U.S. Pat. No. 6,365,373);truncated human growth factor receptor (detected with MAb); mutant humandihydrofolate reductase (DHFR; fluorescent MTX substrate available);secreted alkaline phosphatase (SEAP; fluorescent substrate available);human thymidylate synthase (TS; confers resistance to anti-cancer agentfluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1;conjugates glutathione to the stem cell selective alkylator busulfan;chemoprotective selectable marker in CD34+cells); CD24 cell surfaceantigen in hematopoietic stem cells; human CAD gene to confer resistanceto N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1(MDR-1; P-glycoprotein surface protein selectable by increased drugresistance or enriched by FACS); human CD25 (IL-2α; detectable byMab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable bycarmustine); and Cytidine deaminase (CD; selectable by Ara-C).“Selective medium” as used herein refers to cell growth medium to whichhas been added a chemical compound or biological moiety that selects foror against selectable markers.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” refer to any locus in vitro or in vivo, or in a nucleicacid (e.g., genome) of a cell or population of cells, in which a changeof at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The target sequence can be a genomic locus orextrachromosomal locus.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like. As usedherein, the phrase “engine vector” comprises a coding sequence for anuclease to be used in the nucleic acid-guided nuclease systems andmethods of the present disclosure. The engine vector may also comprise,in a bacterial system, the λ Red recombineering system or an equivalentthereto. Engine vectors also typically comprise a selectable marker. Asused herein the phrase “editing vector” comprises a donor nucleic acid,optionally including an alteration to the target sequence that preventsnuclease binding at a PAM or spacer in the target sequence after editinghas taken place, and a coding sequence for a gRNA. The editing vectormay also comprise a selectable marker and/or a barcode. In someembodiments, the engine vector and editing vector may be combined; thatis, the contents of the engine vector may be found on the editingvector. Further, the engine and editing vectors comprise controlsequences operably linked to, e.g., the nuclease coding sequence,recombineering system coding sequences (if present), donor nucleic acid,guide nucleic acid, and selectable marker(s).

Editing in Nucleic Acid-Guided Nuclease Genome Systems

Nucleic acid-guided nucleases have been used to engineer the genomes ofdiverse organisms; however, differences in intrinsic DNA cuttingactivity, protein expression levels, cellular toxicity and activity indifferent organisms remain significant challenges that necessitates thescreening of many candidate enzymes for editing in each organism.Nucleic acid-guided nucleases with demonstrated activity in vitro and/orin vivo in bacteria, fungi, or mammalian cells are therefore of greatutility. The present disclosure provides a novel catalytically inactiveMAD7 enzyme (dMAD7) that retains the ability to bind DNA in asequence-specific manner. The MAD7 nuclease from which the dMAD7 hasbeen derived was isolated from Eubacterium rectale (MAD7 nuclease aminoacid sequence is SEQ ID NO: 1) (see, e.g., U.S. Pat. Nos. 9,982,279 and10,337,028). The catalytically inactive MAD7 enzyme has the amino acidsequence of SEQ ID NO: 2. In some aspects, the dMAD7 binds to abacterial genome. In other aspects, the dMAD7 binds to a yeast genome.In yet other aspects, the dMAD7 binds to the genome of mammalian cells.

Typically, a nucleic acid-guided nuclease complexed with an appropriatesynthetic guide nucleic acid in a cell can cut the genome of the cell ata desired location. The guide nucleic acid helps the nucleic acid-guidednuclease recognize and cut the DNA at a specific target sequence. Bymanipulating the nucleotide sequence of the guide nucleic acid, thenucleic acid-guided nuclease may be programmed to target any DNAsequence for cleavage as long as an appropriate protospacer adjacentmotif (PAM) is nearby.

The novel MAD-series nucleases may be delivered to cells to be edited asa polypeptide; alternatively, a polynucleotide sequence encoding thenovel MAD-series nuclease(s) is transformed or transfected into thecells to be edited. The polynucleotide sequence encoding the novelMAD-series nuclease may be codon optimized for expression in particularcells, such as archaeal, prokaryotic or eukaryotic cells. Eukaryoticcells can be yeast, fungi, algae, plant, animal, or human cells.Eukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, mouse, rat,rabbit, dog, or non-human mammals including non-human primates. Thechoice of the novel MAD-series nuclease to be employed depends on manyfactors, such as what type of edit is to be made in the target sequenceand whether an appropriate PAM is located close to the desired targetsequence. The novel MAD-series nuclease may be encoded by a DNA sequenceon a vector (e.g., the engine vector) and be under the control of aconstitutive or inducible promoter. In some embodiments, the sequenceencoding the nuclease is under the control of an inducible promoter, andthe inducible promoter may be separate from but the same as an induciblepromoter controlling transcription of the guide nucleic acid; that is, aseparate inducible promoter may drive the transcription of the nucleaseand guide nucleic acid sequences but the two inducible promoters may bethe same type of inducible promoter (e.g., both are pL promoters).Alternatively, the inducible promoter controlling expression of thenuclease may be different from the inducible promoter controllingtranscription of the guide nucleic acid; that is, e.g., the nuclease maybe under the control of the pBAD inducible promoter, and the guidenucleic acid may be under the control of the pL inducible promoter.

In general, a guide nucleic acid (e.g., gRNA), also called a CRISPR RNA(e.g., crRNA), complexes with a compatible nucleic acid-guided nucleaseand can then hybridize with a target sequence, thereby directing thenuclease to the target sequence. The gRNA may be encoded by a DNAsequence on a polynucleotide molecule such as a plasmid, linearconstruct, or the coding sequence may reside within an editing cassetteand is under the control of a constitutive promoter, or, in someembodiments, an inducible promoter as described below.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length.

In the present methods and compositions, the guide nucleic acid isprovided as a sequence to be expressed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript under the control of a promoter, and in some embodiments, aninducible promoter. The guide nucleic acid can be engineered to target adesired target sequence by altering the guide sequence so that the guidesequence is complementary to a desired target sequence, thereby allowinghybridization between the guide sequence and the target sequence. Ingeneral, to generate an edit in the target sequence, the gRNA/nucleasecomplex binds to a target sequence as determined by the guide RNA, andthe nuclease recognizes a proto spacer adjacent motif (PAM) sequenceadjacent to the target sequence. The target sequence can be anypolynucleotide endogenous or exogenous to a prokaryotic or eukaryoticcell, or in vitro. For example, the target sequence can be apolynucleotide residing in the nucleus of a eukaryotic cell. A targetsequence can be a sequence encoding a gene product (e.g., a protein) ora non-coding sequence (e.g., a regulatory polynucleotide, an intron, aPAM, or “junk” DNA).

The guide nucleic acid may be part of an editing cassette that encodesthe donor nucleic acid. Alternatively, the guide nucleic acid may not bepart of the editing cassette and instead may be encoded on the engine orediting vector backbone. For example, a sequence coding for a guidenucleic acid can be assembled or inserted into a vector backbone first,followed by insertion of the donor nucleic acid in, e.g., the editingcassette. In other cases, the donor nucleic acid in, e.g., an editingcassette can be inserted or assembled into a vector backbone first,followed by insertion of the sequence coding for the guide nucleic acid.In yet other cases, the sequence encoding the guide nucleic acid and thedonor nucleic acid (inserted, for example, in an editing cassette) aresimultaneously but separately inserted or assembled into a vector. Inyet other embodiments, the sequence encoding the guide nucleic acid andthe sequence encoding the donor nucleic acid are both included in theediting cassette.

The target sequence is associated with a PAM, which is a shortnucleotide sequence recognized by the gRNA/nuclease complex. The precisePAM sequence and length requirements for different nucleic acid-guidednucleases vary; however, PAMs typically are 2-7 base-pair sequencesadjacent or in proximity to the target sequence and, depending on thenuclease, can be 5′ or 3′ to the target sequence. Engineering of thePAM-interacting domain of a nucleic acid-guided nuclease may allow foralteration of PAM specificity, improve fidelity, or decrease fidelity.In certain embodiments, the genome editing of a target sequence bothintroduces a desired DNA change to a target sequence, e.g., the genomicDNA of a cell, and removes, mutates, or renders inactive a proto-spacermutation (PAM) region in the target sequence. Rendering the PAM at thetarget sequence inactive precludes additional editing of the cell genomeat that target sequence, e.g., upon subsequent exposure to a nucleicacid-guided nuclease complexed with a synthetic guide nucleic acid inlater rounds of editing. Thus, cells having the desired target sequenceedit and an altered PAM can be selected using a nucleic acid-guidednuclease complexed with a synthetic guide nucleic acid complementary tothe target sequence. Cells that did not undergo the first editing eventwill be cut rendering a double-stranded DNA break, and thus will notcontinue to be viable. The cells containing the desired target sequenceedit and PAM alteration will not be cut, as these edited cells no longercontain the necessary PAM site and will continue to grow and propagate.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid. In some embodiments, the donor nucleic acid is onthe same polynucleotide (e.g., editing vector or editing cassette) asthe guide nucleic acid and may be (but not necessarily) under thecontrol of the same promoter as the guide nucleic acid (e.g., a singlepromoter driving the transcription of both the guide nucleic acid andthe donor nucleic acid). The donor nucleic acid is designed to serve asa template for homologous recombination with a target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 20, 25, 50, 75, 100,150, 200, 500, or 1000 nucleotides in length. In certain preferredaspects, the donor nucleic acid can be provided as an oligonucleotide ofbetween 20-300 nucleotides, more preferably between 50-250 nucleotides.The donor nucleic acid comprises a region that is complementary to aportion of the target sequence (e.g., a homology arm). When optimallyaligned, the donor nucleic acid overlaps with (is complementary to) thetarget sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90or more nucleotides. In many embodiments, the donor nucleic acidcomprises two homology arms (regions complementary to the targetsequence) flanking the mutation or difference between the donor nucleicacid and the target template. The donor nucleic acid comprises at leastone mutation or alteration compared to the target sequence, such as aninsertion, deletion, modification, or any combination thereof comparedto the target sequence.

Often the donor nucleic acid is provided as an editing cassette, whichis inserted into a vector backbone where the vector backbone maycomprise a promoter driving transcription of the gRNA and the codingsequence of the gRNA, or the vector backbone may comprise a promoterdriving the transcription of the gRNA but not the gRNA itself. Moreover,there may be more than one, e.g., two, three, four, or more guidenucleic acid/donor nucleic acid cassettes inserted into an enginevector, where each guide nucleic acid is under the control of separatedifferent promoters, separate like promoters, or where all guide nucleicacid/donor nucleic acid pairs are under the control of a singlepromoter. In some embodiments—such as embodiments where cell selectionis employed—the promoter driving transcription of the gRNA and the donornucleic acid (or driving more than one gRNA/donor nucleic acid pair) isan inducible promoter. Inducible editing is advantageous in thatsingulated cells can be grown for several to many cell doublings beforeediting is initiated, which increases the likelihood that cells withedits will survive, as the double-strand cuts caused by active editingare largely toxic to the cells. This toxicity results both in cell deathin the edited colonies, as well as a lag in growth for the edited cellsthat do survive but must repair and recover following editing. However,once the edited cells have a chance to recover, the size of the coloniesof the edited cells will eventually catch up to the size of the coloniesof unedited cells. See, e.g., U.S. Ser. No. 16/399,988, filed 30 Apr.2019; U.S. Ser. No. 16/454,865, filed 26 Jun. 2019; and U.S. Ser. No.16/540,606, filed 14 Aug. 2019. Further, a guide nucleic acid may beefficacious directing the edit of more than one donor nucleic acid in anediting cassette; e.g., if the desired edits are close to one another ina target sequence.

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

In addition, the editing cassette may comprise a barcode. A barcode is aunique DNA sequence that corresponds to the donor DNA sequence such thatthe barcode can identify the edit made to the corresponding targetsequence. The barcode typically comprises four or more nucleotides. Insome embodiments, the editing cassettes comprise a collection of donornucleic acids representing, e.g., gene-wide or genome-wide libraries ofdonor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid isassociated with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furtherencodes a novel MAD-series nuclease comprising one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the novelnuclease comprises NLSs at or near the amino-terminus, NLSs at or nearthe carboxy-terminus, or a combination.

The engine and editing vectors comprise control sequences operablylinked to the component sequences to be transcribed. As stated above,the promoters driving transcription of one or more components of thenovel MAD-series nuclease editing system may be inducible, and aninducible system is likely employed if selection is to be performed. Anumber of gene regulation control systems have been developed for thecontrolled expression of genes in plant, microbe, and animal cells,including mammalian cells, including the pL promoter (induced by heatinactivation of the CI857 repressor), the pBAD promoter (induced by theaddition of arabinose to the cell growth medium), and the rhamnoseinducible promoter (induced by the addition of rhamnose to the cellgrowth medium). Other systems include the tetracycline-controlledtranscriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (PaloAlto, Calif.); Bujard and Gossen, PNAS, 89(12): 5547-5551 (1992)), theLac Switch Inducible system (Wyborski et al., Environ Mol Mutagen,28(4): 447-58 (1996); DuCoeur et al., Strategies 5(3): 70-72 (1992);U.S. Pat. No. 4,833,080), the ecdysone-inducible gene expression system(No et al., PNAS, 93(8): 3346-3351 (1996)), the cumate gene-switchsystem (Mullick et al., BMC Biotechnology, 6: 43 (2006)), and thetamoxifen-inducible gene expression (Zhang et al., Nucleic AcidsResearch, 24: 543-548 (1996)) as well as others.

Typically, performing genome editing in live cells entails transformingcells with the components necessary to perform nucleic acid-guidednuclease editing. For example, the cells may be transformedsimultaneously with separate engine and editing vectors; the cells mayalready be expressing the novel MAD-series nuclease (e.g., the cells mayhave already been transformed with an engine vector or the codingsequence for the novel MAD-series nuclease may be stably integrated intothe cellular genome) such that only the editing vector needs to betransformed into the cells; or the cells may be transformed with asingle vector comprising all components required to perform nucleicacid-guided nuclease genome editing.

A variety of delivery systems can be used to introduce (e.g., transformor transfect) nucleic acid-guided nuclease editing system componentsinto a host cell. These delivery systems include the use of yeastsystems, lipofection systems, microinjection systems, biolistic systems,virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acidconjugates, virions, artificial virions, viral vectors, electroporation,cell permeable peptides, nanoparticles, nanowires, exosomes.Alternatively, molecular trojan horse liposomes may be used to delivernucleic acid-guided nuclease components across the blood brain barrier.Of particular interest is the use of electroporation, particularlyflow-through electroporation (either as a stand-alone instrument or as amodule in an automated multi-module system) as described in, e.g., U.S.Pat. Nos. 10,253,316; 10,329,559; 10,323,242; 10,421,959; 10,453,713 and10,851,389.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are cultured underconditions that promote editing. For example, if constitutive promotersare used to drive transcription of the catalytically inactive dMAD7nuclease and/or gRNA, the transformed cells need only be cultured in atypical culture medium under typical conditions (e.g., temperature, CO₂atmosphere, etc.) Alternatively, if editing is inducible—by, e.g.,activating inducible promoters that control transcription of one or moreof the components needed for nucleic acid-guided nuclease editing, suchas, e.g., transcription of the gRNA, donor DNA, dMAD7, or, in the caseof bacteria, a recombineering system—the cells are subjected to inducingconditions.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. §112, ¶6.

We claim:
 1. A catalytically inactive CRISPR nuclease derived from E.rectale MAD7 (dMAD7) having an amino acid sequence SEQ ID NO:
 2. 2. Thecatalytically inactive CRISPR nuclease of claim 1 for sequence-specificbinding a bacteria genome.
 3. The catalytically inactive CRISPR nucleaseof claim 1 for sequence-specific binding a yeast genome.
 4. Thecatalytically inactive CRISPR nuclease of claim 1 for sequence-specificbinding an isolated mammalian cell genome.